Effect of Emersion on Soft-Shell Clam, Mya Arenaria and the Mussel, Mytilus Edulis Seeds in Relation to Development of Vitality Indices

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Aquat. Living Resour. 27, 91–98 (2014) Aquatic c EDP Sciences, IFREMER, IRD 2014 DOI: 10.1051/alr/2014014 Living www.alr-journal.org Resources

Effect of emersion on soft-shell clam, Mya arenaria and the mussel, Mytilus edulis seeds in relation to development of vitality indices

Rachel Picard1, Bruno Myrand2 and Réjean Tremblay1,a 1 Institut des Sciences de la Mer à Rimouski, Université du Québec à Rimouski, 310 allée des Ursulines, Rimouski, Québec, G5L 3A1, Canada 2 Merinov, 107-125 chemin du Parc, Cap-aux-Meules, Québec, G4T 1B3, Canada

Received 16 August 2014; Accepted 4 December 2014

Abstract – Blue mussels (Mytilus edulis) and soft-shell clams (Mya arenaria) are both aquaculture species in east coast of Canada and US shellfish farmers take advantage of the byssal threads production of mussels for suspension culture and the burrowing behaviour of soft-shell clams for enhancement practices. It is important that these animals attach and burrow efficiently to minimize losses during rearing. The aim of this work was to study two potential vitality indices on mussels (23.6±0.1 mm) and clams (22.6±0.1 mm) seeds following various periods of emersion: attachment strength of Mytilus edulis and burrowing ability of Mya arenaria. The effect of emersion on energy content (proteins, lipids, glycogen) was also examined. We observed no significant decrease in the attachment strength of mussels after air exposure for 78 h or in the burrowing efficiency of soft-shell clams after 54 h. Air exposure had no effect on different lipid classes, proteins, or glycogen content in either mussel or clam tissues. The stressful emersion event induced in our study may not have been high enough to induce detectable behavioural responses. This can be explained by the bivalves’ ability to adapt their metabolism to minimize activity during air exposure. In doing so, they do not consume their energy reserves, which are then still available when specimens are reimmersed. Thus mussels are able to efficiently produce byssal threads and clams to burrow into sediments as soon as they are back in the water.

Keywords: Bivalve aquaculture / Stress indicator / Energy storage / Emersion eﬀect

1 Introduction Attachment and burrowing are two energy-consuming activities that are ecologically relevant and important for suc- Blue mussel (Mytilus edulis) and soft-shell clam (Mya cessful culture production. The byssal thread production is of- arenaria) are two bivalve species used in aquaculture activ- ten considered to be costly in terms of energy requirements ities. Mussel farming began in Canada in the late seventies (Thieltges and Buschbaum 2007; Zardi et al. 2007). Hawkins and is based on submersible longlines to avoid damage from and Bayne (1985) reported that byssal threads can require winter ice cover (Mallet and Myrand 1995). Mussels are the about 8% of the mussel’s total energy costs as well as high most important cultivated bivalve on the east coast of Canada proportions of its carbon (44%) and nitrogen (21%) budgets (Statistic Canada 2010). There is also a strong market potential during summer. Moeser et al. (2006) suggested that the en- for soft-shell clams, thus a growing interest in its cultivation ergetic shifts to reproduction could explain the changes ob- (Pariseau et al. 2007). Mussel and clam growers take advan- served in the mechanical properties of the byssal threads. This tage of the behavioural characteristics of these organisms to hypothesis have been confirmed by others studies showing develop farming techniques. Mussels secrete byssal threads to that spawning influences the attachment strength of mussels attach themselves to substrate. Soon after sleeving (the opera- (Lachance et al. 2008; Seguin-Heine et al. 2014), because tion by which seed mussels are loaded into the mesh sleeves; such stressful event for the mussels could weaken the fibers Mallet and Myrand 1995), spat attach to the rope and mesh by the production of thinner threads (Hennebicq et al. 2013) sleeves on which they will grow until harvest. In contrast, soft- or a decrease of the byssogenesis (Babarro and Reiriz 2010). shell clams are endobenthic: after seeding, clam seed must Babarro and Carrington showed also that mussels (Mytilus gal- burrow into the substrate where they will grow until harvest loprovincialis) changed their energy allocation in relation to (Pariseau et al. 2007). their habitat, as mussels at wave exposed sites allocated resources to reduce risk of dislodgment (smaller and thicker a Corresponding author: [email protected]

Article published by EDP Sciences 92 R. Picard et al.: Aquat. Living Resour. 27, 91–98 (2014)

Table 1. Length and mass of experimental seed used (mean ± SE). Wet mass Total length Species Total Digestive gland All other tissues (mm) (g) (g) (g) Mytilus edulis (n = 525) 23.6 ± 0.11.45 ± 0.02 0.077 ± 0.003 0.436 ± 0.007 Mya arenaria (n = 450) 22.6 ± 0.11.43 ± 0.02 0.043 ± 0.001 0.447 ± 0.008 shell, stronger byssal threads) instead of growth and reproduction. However, in Magdalen Islands lagoon, we noted that the energy level for adult suspension-culture mussels was not a limiting factor in terms of byssal production (Lachance et al. 2011). Burial involves muscular movements requiring energy (Pariseau et al. 2007). Indeed, mussels have to attach to sleeves to avoid slippage (fall-off) to the bottom while clams have to burrow rapidly to avoid benthic predators as well as dislodge- ment by waves and currents (Hunt and Mullineaux 2002). In their natural intertidal habitat, mussels and clams undergo periodic air exposure and are well adapted to such environmental conditions. Mussels have the ability to hermetically close their valves to avoid desiccation. soft-shell clams are not as threat- ened as mussels by desiccation because the substrate in which they burrow never completely dries, thus providing them a per- manently wet habitat. Their valves do not close hermetically, but they will withdraw their siphons into the shell. During aquaculture operation, this emersion periods could be more important than the periodic air exposure undergo in natural environment and combined with manipulation, these stressful emersion event could have a significant energetic impact on clams and mussels (Tremblay and Pellerin-Massicotte 1997; Fig. 1. Sampling sites for Mytilus edulis (Havre-aux-Maisons) and Leblanc et al. 2005; Babarro et al. 2007;Brenneretal.2012) Mya arenaria (Dune du Nord) seed in the Magdalen Islands, Québec, Canada. and eventually impact the mussels’ byssogenesis and clams’ burrowing behaviour. ff The aim of this work was to test the e ects of extended Islands (Québec, Canada) (Table 1,Fig.1). These young mus- air exposure with 100% humidity on the attachment capacity sels and clams are generally immature in May in this area of young blue mussels and on the burrowing ability of young (Rodhouse et al. 1986; Brulotte and Giguère 2007), so there soft-shell clams. We hypothesized that attachment and burial was no energy invested in gonadal development. All specimens could be reduced due to the higher energy investment needed were placed in flow-through tanks for three days to recover to maintain basal metabolism when faced with the stressful from harvesting operations; tanks were supplied with sand- emersion event. Even though bivalves can switch from aerobic filtered water pumped from the nearby Havre-aux-Maisons to anaerobic metabolism to limit energy demands during emer- Lagoon. After this period, 600 specimens of each species were sion (Shick and Widdows 1981;Newell1991), the by-products brought into a temperature-controlled room (10 ◦C) and placed of anaerobic metabolism accumulated during emersion are ex- between two layers of brown paper saturated with seawater to pulsed by overshooting filtering (Newell 1991) when animals avoid tissue desiccation. Air exposure treatments lasted 6, 24, are returned to the water. Due to these energetic costs, mussels 30, 48, 54, and 78 h for mussels and 6, 24, 30, 48, and 54 h might produce weaker threads while soft-shell clams might for soft-shell clams. Every morning, the paper was sprayed ff take longer to bury completely. The e ect of reimmersion after with seawater to maintain humidity at 100%. Control mussels emersion on energy storage was also studied in the digestive and clams representing air exposure of 0 h, were left in the gland and in all other tissues combined. acclimation tank until the behavioural experiments.

2 Materials and methods 2.2 Tank conditions

2.1 Collection and emersion of mussels and clams We used 3 tanks (1.9 m long × 1.1 m wide × 0.6 m high), each supplied with seawater at a ﬂow of 10 L min−1. In late May 2007 (seawater temperature of 10 ◦C), seeds The water level was maintained at 30 cm above the bot- of blue mussels were sampled from longlines and soft-shell tom, and lights above the tanks were on during all experi- clams were manually collected at low tide from a natural pop- ments. Water salinity and temperature were measured in ex- ulation both in Havre-aux-Maisons Lagoon from the Magdalen perimental tanks at the beginning, middle, and end of each R. Picard et al.: Aquat. Living Resour. 27, 91–98 (2014) 93 experiment with a YSI-85 thermosalinometer (Yellow Springs mussels, each clam was dissected and the digestive gland and Instruments, OH, USA). Triplicate samples were taken in each remaining tissues were separated. Wet tissue was weighed and of the 3 experimental tanks at the beginning and at end of each kept at −80 ◦C until biochemical analyses. experiment for total particulate organic matter (TPM) analysis.

2.4 Biochemical analysis 2.3 Behavioural analysis For each species, the digestive gland and remaining tis- Mussels (n = 75 per treatment) were randomly chosen for sues of 12 specimens by treatment were randomly sampled and the seven emersion periods (0, 6, 24, 30, 48, 54, and 78 h) analyzed individually to compare the two treatments: control and two hours prior the reimmersion, each mussel was glued (no emersion) vs. 78 h of emersion following by 22 reimmer- (Bostik cyanoacrylate adhesive glue) onto an individual plas- sion for mussels and control vs. 54 h of emersion for soft-shell tic plate by its right valve (ventral edge facing down). Each clams. Proteins were determined by colorimetry with a spec- individual plastic plate with its glued mussel was then perpen- trophotometer (Molecular Devices Vmax Kinetic Microplate dicularly inserted into one of the five grooves of a larger main Reader, USA) according to Bradford (1976). Lipid classes (tri- plastic plate. Each groove could hold five individual plastic acylglycerols [TAG], sterols [ST], and phospholipids [PL]) plates. This system allowed the mussels to attach their newly were determined by chromarod thin-layer chromatography secreted byssal threads to the main plate. Each plate with its coupled with an Iatroscan flame ionization detection system mussel was subsequently pulled away to measure the mussel’s (Iatroscan MK-6) according to Parrish (1987). Because of the attachment strength to the main plate without interfering with microquantities of tissues, it was impossible to use the same the attachment of its neighbours (see below). Each main plate specimen’s tissues for all biochemical analyses. Thus, glyco- measured 60 cm long × 60 cm wide × 1 cm thick and held gen analyses were made on 5 different specimens randomly 25 individual plates arranged vertically. The individual plates chosen per tank and per emersion period and determined were 10 cm long × 5cmwide× 1 cm thick. The plate sys- by colorimetry and spectrophotometry (Spectrophotometer tems were placed at the bottom of each of the three tanks for Beckman DU 640) according to Carr and Neff (1984). reimmersion for 22 h. Long pieces of stainless steel were put on each row of individual plastic plates to keep the plate sys- tems at the bottom. Water was distributed along each row of 2.5 Statistical analysis the main plate via a perforated pipe. After 22 h of reimmersion, the attachment strength of individual mussels was de- Mussel behaviour experiments were run in triplicate (three termined. A small hook joined by string to an endless screw different tanks), with 25 mussels per emersion period. The was attached to an individual mussel plate and connected to percentage of totally buried clams was calculated for each a digital force gauge. Mussel plates were drawn up mechani- tank, thus providing triplicate values for each emersion period. cally at constant low speed by a small motor until the plate dis- 20 completely buried clams were randomly chosen to quan- lodged, as described by Lachance et al. (2008). The minimum tify the burrowing activity. For biochemical analysis, 5 mus- force required to dislodge a plate with its glued mussel was sels and clams per tank (replicate) per emersion period were recorded with a 10 N digital force gauge (Q-graph QuantrolTM analyzed. For behavioural analysis, one-way ANOVAs were software; Dillon, Fairmont, MN, USA). The force needed to used to compare the effect of the emersion period on the at- dislodge each plate with no mussel had been recorded before- tachment strength of mussels and on the percentage of clams hand and was subtracted from the force measured during the completely buried and the burrowing initiation time. Factorial experiment to eliminate the individual plate weight. The re- ANOVAs (main factors: emersion duration and tissue) were sulting value is the net attachment strength. After attachment used for biochemical analyses. Tukey tests were applied af- strength measurements, shell length was determined with an ter Bonferroni corrections if assumption of ANOVA were met. electronic caliper (Fowler Sylvac) and total wet weight was When needed, data were transformed (log10 or square root) measured. Each mussel was then dissected and the tissues to respect test conditions. Residual distribution normality was separated into digestive gland and remaining tissues. Tissues analyzed with a Kolmogorov-Smirnov non-parametric test and were weighed separately and kept at −80 ◦C for subsequent homogeneities of variance were determined with Levene’s test. biochemical analyses. All statistical analyses were performed using Systat 11 (Systat Before reimmersion of soft-shell clams, three trays of ap- Software Inc., San Jose CA, USA). proximately 70 cm long × 40 cm wide × 20 cm high were filled with pre-sifted (800 µm) medium sand and placed in the middle of each of the three experimental tanks. Twenty-five clams 3 Results (n = 75 per treatment) were placed in five rows of five clams in each tray (triplicates) for each air exposure trial (0, 6, 24, 30, 3.1 Water analysis 48, 54 h). At this point, the degree of burial (not buried at all, 50% buried, or completely buried) was recorded every 15 min Temperature and salinity were similar for all treatments for four hours. The time required for clams to start burrowing for both species. Mean water temperature in the 3 tanks dur- and the percentage of clams completely buried were recorded ing the experiment with mussels was 10.3 ± 0.1 ◦C while after four hours of reimmersion. After burial measurements, salinity was 31.17 ± 0.03. Concentrations of total particulate shell length and total weight were measured. As was done with matter (TPM) and particulate organic matter (POM) during 94 R. Picard et al.: Aquat. Living Resour. 27, 91–98 (2014)

1.4 120 df=6, F=4.859, p=0.007, n=525 A df=5, F=0.33, p=0.885, n=450 B df=5, F=2.044, p=0.125, n=450 C 50

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0.0 0 0 0 1020304050607080 0 1020304050607080 0 1020304050607080 Emersion time (h) Emersion time (h) Emersion time (h) Fig. 2. Attachment strength of mussels (A), percentage of totally burrowed clams (B), and time required to initiate burrowing in clams (C), for diﬀerent lengths of time (mean ± SE) exposed to air (10 ◦C and 100% humidity), 0 represent the control always submerged in seawater.

Table 2. Biochemical variables analyzed in Mytilus edulis and Mya arenaria digestive gland and all the other tissues (mean ± SE). Concentrations were pooled when no significant difference between tissues was found. TAG: triacyglycerols, ST: sterols, PL: phospholipids. Variable Digestive gland All other tissues %TAG 55.7 ± 7.224.1 ± 2.6 %ST 3.0 ± 0.66.7 ± 0.4 %PL 22.0 ± 3.946.9 ± 2.0 Mytilus edulis TAG/PL 43.4 ± 11.02.3 ± 0.2 TAG/ST 5.1 ± 1.90.7 ± 0.1 Glycogen (µgg−1*) 33.7 ± 4.1 Proteins (mg g−1*) 6.6 ± 0.38.6 ± 0.6 %TAG 17.4 ± 1.88.4 ± 0.9 %ST 5.4 ± 0.49.9 ± 0.4 %PL 42.3 ± 2.065.3 ± 3.2 Mya arenaria TAG/PL 8.4 ± 0.7 TAG/ST 3.4 ± 0.50.9 ± 0.1 Glycogen (µgg−1*) 10.6 ± 1.4 Proteins (mg g−1*) 4.4 ± 0.3 ∗ Pergramofwetmass. this experiment were respectively 0.60 ± 0.14 mg L−1 and significantly different after emersion of 6 to 54% with an over- 0.51 ± 0.01 mg L−1. During the experiment with clams, mean all mean of 86 ± 2% (Fig. 2B). The time required for clams water temperature was 12.8 ± 0.2 ◦C, salinity 31.04 ± 0.03, to initiate burial was not related to the duration of air ex- TPM 0.84 ± 0.10 mg L−1 and POM 0.38 ± 0.06 mg L−1. posure and started after 30 min in all emersion treatments (Fig. 2C). Less than 5% of clams do not burrow during all the experiment. 3.2 Behavioural analysis 3.3 Biochemical analysis Behavioural analyses have been realized on seed around 23 mm for both species (Table 1). For Mytilus edulis,airex- There was no significant effect of emersion or emersion du- posure had a significant effect on their attachment strength ration on the various biochemical parameters for either species comparatively to control (mussels without emersion, Fig. 2A). (Tables 2 and 3). However, we did find differences in the con- Mussels emerged from 6 to 54 h and reimmerged for 22 h centrations of the most biochemical constituents in the two were attached significantly more strongly (nearly twice) to the tissue groups examined (Tables 2 and 3). plate compared to control mussels (no emersion at all, Tukey In mussels, biochemical contents in the digestive gland p < 0.05). The mean strength measured in control animals was were significantly different from those found in the remain- 0.64 N while mussels showing maximal value (48 h emersion) ing tissues except for the glycogen (Table 3). TAG percentage showed mean attachment strength of 1.08 N. and the TAG/PL and TAG/ST ratios were higher in the diges- For Mya arenaria, the percentage of individuals per tank tive gland while % ST, % PL, and protein content were higher that were completely buried after 4 h of reimmersion was not in the remaining tissues (Table 2). R. Picard et al.: Aquat. Living Resour. 27, 91–98 (2014) 95

Table 3. Results of factorial ANOVA examining the effects of emersion time and tissues on the biochemical constituents (TAG: triacylglycerols, ST: sterols, PL: phospholipids, ratio between TAG and ST, ratio between TAG and PL, proteins and glycogen) of mussels (Mytilus edulis)and soft-shell clams (Mya arenaria). Bold indicated significant effect of factors. TAG, ST and PL were tested on % of total lipids and proteins and glycogen on concentrations in mg g−1 and ug g−1 respectively (n = 12). Mya arenaria Mytilus edulis Variables Factors df F p df F p Emersion 1 0.031 0.865 1 0.011 0.919 %TAG Tissues 1 14.107 0.006 1 16.451 0.004 Emersion*Tissues 1 0.215 0.655 1 0.067 0.803 Emersion 1 0.000 0.993 1 2.146 0.181 %ST Tissues 1 22.144 0.002 1 55.595 0.001 Emersion*Tissues 1 0.711 0.424 1 0.001 0.980 Emersion 1 0.055 0.820 1 0.482 0.507 %PL Tissues 1 26.271 0.001 1 33.738 0.001 Emersion*Tissues 1 0.084 0.779 1 0.774 0.405 Emersion 1 0.376 0.557 1 0.173 0.689 TAG/ST Tissues 1 8.750 0.018 1 22.827 0.001 Emersion*Tissues 1 0.069 0.799 1 0.008 0.929 Emersion 1 0.025 0.877 1 1.866 0.209 TAG/PL Tissues 1 14.197 0.005 1 2.228 0.174 Emersion*Tissues 1 0.032 0.862 1 0.024 0.881 Emersion 1 0.673 0.436 1 4.448 0.068 Proteins Tissues 1 8.556 0.019 1 0.068 0.800 Emersion*Tissues 1 0.508 0.496 1 0.579 0.468 Emersion 1 0.131 0.726 1 1.313 0.285 Glycogen Tissues 1 3.223 0.110 1 5.264 0.051 Emersion*Tissues 1 0.091 0.770 1 0.112 0.747

Except for the TAG/PL ratio, protein content, and glyco- ment strength and burrowing) are not good indicators of vi- gen, the energy components of clams were different between tality for mussels and clams or that the stress used (emersion the two groups of tissue (Table 3). TAG percentage and for the facility to control the intensity) was not appropriate to the TAG/ST ratio were higher in the digestive gland while test the seed vitality. It was also surprising to observe that seed ST and PL percentages were higher in the remaining tissues clams need more that 30 min to initiate burrowing. Generally, (Table 2). healthy seed clams (around 15 mm in shell length) burrow within 10 min (Emerson et al. 1990; Beal and Vencile 2001; Appelbaum et al. 2011). These results could suggest that vital- 4 Discussion ity of clams’ seed in end of May in this area could be too low to observed significant changes. This longer emersion time could In this study, the duration of air exposure (78 h for mus- be also related to higher seed size used in our study (around sels and 54hforclams)hadnonegativeeffect on attachment 22 mm), as Pariseau et al. (2007) observed an inverse relation- of mussels or on burial of clams after reimmersion. Thus, our ship between size and burial level. We observed an increase hypothesis that attachment and burial efficiency are reduced of attachment strength after 6 h emersion in M. edulis seed. by the higher energy investment needed to maintain basal Results on Perna canaliculus, where 4 h of emersion followed metabolism, when faced with the stressful emersion event, was by 20 h of reimmersion reduced significantly the activity of not confirmed. Significant higher attachment strength were ob- seed mussels, the vital staining (heat movements, gill cilia, served from mussels emerged for 6 h and have been main- foot, shell valves, mantle and siphon) and rate of retention esti- tained until 78 h emersion. Our results suggest that emersion mated by byssal thread attachment (Webb and Heasman 2005; periods of 54 h for clams and 78 h for mussels at 10 ◦C Carton et al. 2007). These results could suggest that M. edulis and 100% humidity are not stressful enough to alter these seem largely more resistant to emersion than P. canaliculus. behaviours. In a previous study on Mya arenaria, Pariseau et al. (2007) found a similar result with individuals held out 4.1 Emersion effect of the water for only 4 h in the sun without modification of burial behaviour compared to controls. A possible explanation The capacity of many bivalve species to reduce their en- could be that the behavioural indices examined here (attach- ergy demands during emersion might explain the fact that no 96 R. Picard et al.: Aquat. Living Resour. 27, 91–98 (2014) behavioural differences were observed in mussels and clams event showed lysosomal destabilization, but their metabolic following different emersion periods. In addition to disrupt- rate returned to control levels 12 h after reimmersion. Simi- ing their respiratory activities, air exposure prevents bivalves lar results were observed in intertidal conditions for M. edulis from feeding and might add a desiccation stress. During this and M. arenaria (Tremblay and Pellerin-Massicotte 1997). period, mussels and clams must rely on anaerobic metabolism As was the case with the behavioural responses, the bio- (de Zwaan and Wijsman 1976; Schick et al. 1986; Sobral and chemical results suggest that our experimental emersion treat- Widdows 1997; Babarro et al. 2007), which should lead to ments (time, humidity, and temperature) did not stress blue a marked metabolic depression (Shick and Widdows 1981; mussels or soft-shell clams. However, mussels and clams may Le Moullac et al. 2007). The energy requirements met by have had time to recover when reimmersed for 22 h. It is possi- anaerobiosis are only 4% of normal aerobic metabolism in ble that mussels and clams were stressed by emersion but that the blue mussel (Widdows 1989). During emersion, metabolic their subsequent reimmersion allowed them to return to control activities are thus reduced to a minimum to diminish the ef- levels. In this context, the intensity and duration of the stress fects to this stress until reimmersion. Furthermore, when mus- events could have been insufficient to be measured by the cho- sels are exposed to air, they show a gaping behaviour (peri- sen indicators (energy reserves and behaviour) at the end of the odic opening and closing of the shells), which provides them reimmersion. with a low oxygen renewal in the tissues (Widdows et al. 1979; Guderley et al. 1994). Significant reductions of burial behavioral following exposition to high hypoxia level have been al- 4.2 Tissue effect ready demonstrated for other clams’ species (Long et al. 2008; Lee et al. 2012). During this hypoxia, the metabolism was sup- Differences were observed in most biochemical concentra- ported by anaerobic activity related to a decline of glycogen tions between the two tissue groups depending on the compo- and protein content (Lee et al. 2012). In our results, the ab- nent. TAG percentage was higher in the digestive gland than sence of modification of glycogen and protein use in clams and in the other tissues for both species. TAG is an important en- mussels tissues suggests that the emersion conditions seem not ergy source for bivalves (de Zwaan and Mathieu 1992), and impact energetic reserves. We suggest that emersion in 100% the digestive gland is where digestion, energy storage, and the humidity and 10 ◦C and the potential air gaping and metabolic distribution of metabolic reserves occur. Our results suggest despression, seem sufficient to maintain seed vitality during that mussels and clams had enough energy available to resist 54 h for clams and 78 h for mussels. physiological stress. The high TAG concentration can probably be explained by an accumulation of plankton rich in TAG When reimmersed, mussels increase their respiration rate that were ingested previous to emersion and during the reim- to compensate for the oxygen debt accrued during anaerobic mersion period (Caers et al. 2000). In contrast to TAG, the per- metabolism and start filtering and feeding to rebuilding their centages of structural lipids (ST and PL) were higher in the re- energy stores (Zandee et al. 1986). During the 22 h of reim- maining tissues as already observed in literature (Moreno et al. mersion, energy transfer between mussel digestive gland and 1976;Pernetetal.2007). These tissues use energy from these other tissues probably did not have time to occur: Thompson lipids for growth, which could explain why structural lipids et al. (1974) found that energy transfer between the digestive are more abundant in these tissues than in the digestive gland, gland and other tissues takes about 7–10 days in summer and which is an energy storage organ. fall but up to 35 days in winter, when food is scarce. Thus, the While glycogen concentration was the same for both energy component measured in tissues other than the diges- groups of tissues in the blue mussel, its level in M. arenaria tive gland after reimmersion would reveal the energetic cost digestive gland was about half that found in the remaining tis- of emersion, attachment, and the increased respiration rate re- sue. This might be explained by the fact that glycogen content quired to evacuate the by-products of anaerobic metabolism. increases in M. arenaria mantle tissue when food is abundant Our results show that there was no impact of these three po- (Gabbott and Bayne 1973), suggesting that clams ingested and tentially energy-consuming activities on energy components in stored glycogen before emersion. Even though clams were not non-digestive gland mussel tissues, meaning that animals did supposed to be sexually mature, some small individuals, like not have to consume their energy resources, or at least not to a our individuals around 23 mm, can have limited fertility and detectable degree. reproduce (Brousseau 1978; Roseberry et al. 1991). It is possi- This stability of the behavioural response and in the differ- ble that glycogen was invested in gonadal development in such ent energy components analyzed is in agreement with Harding individuals. et al. (2004), who found that dry storage (emersion) of mussels at high humidity and 2–4 ◦Cforaperiodupto12days followed by a 24 h reimmersion can reduce the stress response 5 Conclusion and provide a better quality product with longer shelf life. In contrast, Almeida et al. (2005) found that emersion of Perna Relatively simple behavioural tests could be very useful to perna for 24 h caused a clear increase in the levels of lipid farmers, providing them with a rapid and inexpensive way to peroxidation and oxidative DNA damage. Nevertheless, when determine the vitality of their seed stock. The aquaculture in- these mussels were reimmersed in seawater for three hours, dustry needs to develop easy and rapid vitality indicators to the levels of damage to lipids and DNA went back to control limit fall-off and losses. Our results suggest that an emersion values. Hole et al. (1995) also found that 2- to 4-year-old blue duration of 2–3 days at 10 ◦C and 100% humidity is proba- mussels exposed to a 24 h hypoxia and hyperthermia (28 ◦C) bly not stressful enough to be measured with the behavioural R. Picard et al.: Aquat. Living Resour. 27, 91–98 (2014) 97 and biochemical indicators chosen. A longer duration, higher Carr R.S., Neff J.M., 1984, Quantitative semi-automated enzy- temperature, or drier environment during emersion, or another matic assay for tissue glycogen. Comp. Biochem. Physiol. 77B, kind of stressful event, may be needed to produce an effect 447–449. on these indicators. Thus, the use of these behavioural indi- Carton A.G., Jeffs A.G., Foote G., Palmer H., Bilton J., 2007, cators for aquaculture purposes needs further investigation. Evaluation of methods for assessing the retention of seed mussels Nevertheless, behavioural indicators are valuable because of (Perna canaliculus) prior to seeding for grow-out. Aquaculture their low cost and ease of use. The behavioural indicators we 262, 521–527. chose are known to be influenced by temperature, salinity, food Clements J.C., Hunt H.L., 2014, Influence of sediment acidification availability, size, reproductive cycle and sediment carbonate and water flow on sediment acceptance and dispersal of juvenile geochemistry (Matthiessen 1960; Lardies et al. 2001; Pariseau soft-shell clams (Mya arenaria L.). J. Exp. Mar. Biol. Ecol. 453, et al. 2007; Lachance et al. 2008; Clements and Hunt 2014). 62–69. Our results show that emersion at low temperature (10 ◦C) de Zwaan, A., Wijsman T.C.M., 1976, Anaerobic metabolism in and high humidity for as long as 78 h for mussels and 54 h Bivalvia (Mollusca): Characteristics of anaerobic metabolism. Comp. Biochem. Physiol. 54B, 313–324. for clams has no effect on the energy reserves and capacity of mussels to produce byssus or of clams to burrow. 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